The etiology of interstitial cystitis (IC) is unknown. The central thesis of this continuation application is that cryptic bacterial genomes in tissue in the form of cell wall-deficient/defective bacteria (CWDB) and/or nutritionally deficient bacteria not cultured by routine methods may be important organisms overlooked in the etiology of IC. Data obtained to date substantiate our hypothesis of bacterial presence in the bladder in the absence of conventional, positive, routine, urine cultures. Utilizing the polymerase chain reaction (PCR), 29% of IC patients showed presence of bacterial 16S rRNA genes in biopsied urinary bladder tissue whereas no amplification was seen in reactions containing input DNA from 9 control patients or in 4 reactions containing no input DNA. Additionally, urine and bladder tissue from all IC patients demonstrated unusual forms in culture, on specialized media, that morphologically resemble CWDB. This grant would expand the number of IC patients from 60 to 164 for detection of bacterial genomes in bladder tissues and from 60 control subjects to 110 during the next five years. These numbers should statistically guarantee with confidence detection of shifts in PCR positivity of about 10-12%. We will identify and characterize the bacteria present in those subjects, as well as the isolated aberrant form(s) and determine the relationship (if any) of these forms to the 16S rRNA genes amplified. PCR amplification and gene sequencing will be used to determine whether there are predominant genera (or species) involved in IC. We will develop in- situ PCR capable of detecting these organisms in section biopsies. Reverse Transcriptase PCR (RT-PCR) will be used as an additional method for analyzing mRNA in tissue since it is a more sensitive alternative to traditional PCR. We will search for an increase in the amount of common bacterial antigens and metabolic products in urine (lipopolysaccharides, enterobacterial common antigen, lipoteichoic and teichoic acids) as an index of metabolically active bacteria. Monoclonal and polyclonal antibodies will be made against the organisms and/or their products for use as diagnostic reagents and for confirming the presence of organisms in tissues and body fluids at the light microscopic and ultrastructural levels. Attempts will be made to establish an IC model in BALB/c mice inoculated with selected bacteria; the course of the disease will be followed by PCR, culture, immunocytochemistry, cytokine detection, T- lymphocyte responses to organisms and effectiveness of antibiotics in clearing infection and inflammation. IC patients with (+) and (-) PCR findings will be treated with antibiotics; the course of the disease will be monitored, clinically, by the immune response, and microbiology of patient specimens. Data generated should allow us to conclude whether bacterial presence in the urinary bladder has an effect on the disease and its resulting morbidity.